Viruses in the family Poxviridae, including vaccinia virus (VACV) and variola virus, are characterized by a large linear double-stranded DNA genome (130-300 kb) packaged in a relatively large virion (xcx9c350xc3x97270 nm), and a cytoplasmic site of replication (reviewed by Moss, 1996, In xe2x80x9cFields Virologyxe2x80x9d, D. M. Knipe et al. Eds., vol. 3, pp 2637-2671. Lippincott-Raven, Philadelphia). Assembly of VACV virions begins with condensation of dense granular material into membrane-wrapped particles called intracellular mature virions (IMV). Recent findings indiate the IMV are wrapped by a single membrane (Hollingshead et al., 1999, J. Virol. 73, 1503-1517) rather than a double membrane as previously reported. IMV are then enveloped in two additional membranes derived from the trans Golgi to form multiple membrane-warpped particles called intracellular enveloped virions (IEV) (Schmelz et al., 1994, J. Virol. 68, 130-147). IEV are moved, possibly by actin polymerization (Cudmore et al., 1995, Nature 378, 636-638), to the cell periphery, where the outermost membrane fuses with the cell plasma membrane, exposing a cell-associated eneveloped virion (CEV) (Blasco and Moss, 1991, J. Virol. 65, 5910-5920). CEV are released from the cell as extracellular enveloped virions (EEV), which play a role in long-range spread of the virus (Payne, 1980, J. Gen. Virol. 50, 89-100). IMV released from disrupted cells and EEV are both infectious forms of VACV.
The current smallpox vaccine (live vaccinia virus) has many drawbacks including: adverse reactions, scarring, ocular autoinoculation, dissemination in immunocompromised persons, and dwindling stocks. Cell culture derived vaccines, are being developed; however, these vaccines are also live viruses and pose many of the same drawbacks that plague the current vaccine. A DNA-based replacement vaccine could conceivably effectively protect against smallpox, other poxviruses, and engineered vaccinia viruses without any of the drawbacks associated with live-virus vaccines. This is especially relevant to immunocompromised persons who cannot be vaccinated with live vaccinia virus.
Naked DNA vaccines have been used to generate protective immune responses against numerous pathogenic agents, including many viruses (Gregoriadis, 1998, Pharmacol. Res. 15, 661-670). In general, naked DNA vaccines involve vaccination with plasmid DNA that contains a gene of interest controlled by a cytomegalovirus (CMV) promoter. When the plasmid is introduced into mammalian cells, cell machinery transcribes and translates the gene. The expressed protein (immunogen) is then presented to the immune system where it can elicit an immune response. One method of introducing DNA into cells is by using a gene gun. This method of vaccination involves using pressurized helium gas to accelerate DNA-coated gold beads into the skin of the vaccinee.
To identify potential targets for poxvirus vaccines or therapeutics, we generated and characterized a panel of VACV-specific monoclonal antibodies (MAbs). Passive protection experiments in mice indicated that neutralizing MAbs binding a 29-kDa protein (e.g., MAb-10F5, MAb-7D11), and nonneutralizing MAbs binding a 23- to 28-kDa protein (e.g., MAb-1G10) protected against challenge with VACV (strain WR). The target of MAb-7D11 was the product of the L1R gene (Wolffe et al. 1995, Virology 211, 53-63), and the target of MAb-1G10 was the product of the A33R gene (Roper et al., 1996, J. Virol. 70, 3753-3762). In this report, the L1R and A33R gene products will be called L1R and A33R, respectively. L1R is an essential myristoylated protein associated with the IMV membrane and is thought to play a role in IMV attachment or penetration (Franke et al., 1990, J. Virol. 64, 5988-5996; Ravanello et al., 1993, J. Gen. Virol. 75, 1479-1483; Ichihashi et al., 1994, Virology 202, 834-843; Ravanello and Hruby, 1994, J. Gen. Virol. 75, 1479-1483; Wolffe et al., 1995, supra). A33R is a nominally nonessential glycosylated/palmitated protein that forms dimers and is incorporated into the outer membrane of EEV (Payne, 1992, Virology 187, 251-260; Roper et al., 1996, supra). A33R is thought to be involved in facilitating direct cell-to-cell spread via actin-containing microvilli (Roper et al., 1998, J. Virol. 72, 4192-4204). Homologs of L1R and A33R are present in other Orthopoxviruses, e.g. between VACV and variola, L1R identity is 99.6% and A33R is 94.1% (Massung et al., 1994, Virology 201, 215-240).
To determine whether vaccination with the L1R, encoding an IMV immunogen and/or A33R, encoding an EEV immunogengene could elicit protective immunity, we constructed plasmids expressing either L1R or A33R under control of the CMV promoter and tested these plasmids, and combinations of these plasmids, for immunogenicity and protective efficacy in mice. Our results indicated that vaccination with both L1R and A33R proteins, when loaded on different gold beads and hence delivered to different cells, was more effective than vaccination with either protein by itself or vaccination with L1R and A33R on the same particle.
Furthermore, our data indicates that a composition consisting of a combination of vaccinia IMV and EEV immunogens would provide a better vaccine protective against two infectious forms of vaccinia. Thus, this invention could serve to replace the existing vaccine, and could serve to vaccinate the subpopulation that cannot be vaccinated with a live virus vaccine.
In this report, we describe a new recombinant DNA vaccine approach that involves vaccination with naked DNA expressing individual poxvirus cDNAs. Naked DNA vaccination involves delivery of plasmid DNA constructs with a gene(s) of interest into the tissue of the vaccinee (reviewed in Robinson and Torres, 1997, Semin. Immunol. 9, 271-283; and Gregoriadis, 1998, Pharm. Res. 15, 661-670). The gene(s) of interest is controlled by a mammalian or virus promoter (e.g., the cytomegalovirus immediate early promoter) that facilitates expression of the naked DNA gene product(s) within the vaccinee""s cells. This intracellular expression can elicit both humoral and cell-mediated immune responses (Robinson and Torres, 1997, supra; and Gregoriadis, 1998, supra). Methods of DNA delivery include needle inoculation, oral or pulmonary delivery, and inoculation by particle bombardment (i.e., gene gun). DNA vaccination by each of these methods elicits protective immunity against many different pathogens including numerous viruses (Robinson and Torres, 1997,supra; and Gregoriadis, 1998, supra).
In this report, we demonstrate that naked DNA vaccination with a combination of IMV and EEV immunogens, for example, L1R and/or A33R, respectively, elicits poxvirus-specific antibody responses in rodents. More importantly, we demonstrate that DNA vaccination with the L1R and A33R elicits neutralizing antibodies and protects mice against a lethal poxvirus infection.
Therefore, it is one object of the present invention to provide a poxvirus DNA vaccine comprising a poxvirus cDNA. More specifically, the present invention relates to a poxvirus DNA vaccine comprising genes found in the intracellular mature form of the virus (IMV) for example, L1R and A27L in combination with genes found in the extracellular enveloped form of the virus (EEV) for example, A33R and B5R. The vaccine may consist of preferably one gene from IMV and one from EEV, more preferably, the vaccine may consist of three or four genes where at least one gene is from EEV and one is from IMV.
It is another object of the present invention to provide a method for eliciting in a subject an immune response against poxvirus, the method comprising administering to a subject a DNA fragment comprising a poxvirus cDNA. More specifically, the present invention relates to a method for eliciting an immune response against poxvirus by providing an IMV gene and an EEV gene.
In one aspect of the invention, the DNA vaccine is delivered by coating a small carrier particle with the DNA vaccine and delivering the DNA-coated particle into an animal""s epidermal tissue via particle bombardment. This method may be adapted for delivery to either epidermal or mucosal tissue, or delivery into peripheral blood cells, and thus may be used to induce humoral, cell-mediated, and secretory immune reponses in the vaccinated individual. In one aspect of the invention, the IMV and EEV cDNA is delivered by coating different carrier particles and not combined on one carrier particle.
The DNA vaccine according to the present invention is inherently safe, is not painful to administer, and should not result in adverse side effects to the vaccinated individual. In addition, the invention does not require growth or use of poxvirus, which may be spread by aerosol transmission.